Project Summary/Abstract:FDA-approved prophylactic human papillomavirus (HPV) vaccinesare unable to clear pre-existing HPV infections, the primary etiologic factor for subsets of anogenital and oropharyngeal cancers, including essentially all cervical cancers. Therefore, therapeutic HPV vaccines are being developed andevaluatedfortheirabilitytogenerateHPV-specificCD8+TcellresponsestoclearHPVantigen-expressing infectedormalignantcells.Forexample,TA-CINisarecombinantHPV16E6,E7,andL2fusionproteinvaccine that can generate both HPV16 E6/E7-specific CD8+ T cell responses as well as L2-specific antibodies. Unfortunately, due to its low intrinsic immunogenicity, TA-CIN has lacked efficacy to date in clinical settings against HPV lesions, despite being well tolerated by vaccinated patients. FMS-like tyrosine kinase 3 ligand (Flt3L)isacytokinethathasthepotentialtoenhancetheimmunogenicityofTA-CINvaccinationduetoitscentral roleinstimulatingtheexpansionanddifferentiationofCD8+andCD103+dendriticcells(DCs)thatplayacrucial role in antigen cross-presentation for the activation of CD8+ T cell responses, including following vaccination. However, the efficacy of Flt3L treatment is limited by its lack of targeting and short half-life in vivo. We have overcome these disadvantages of Flt3L by fusing it to albumin (Alb), resulting in the development of a novel fusionproteinadjuvant,Alb-Flt3L.UtilizingtheFcreceptor(FcRn)-mediatedinvivohalf-lifeprolongingandthe lymph node targeting effects of the Alb fusion, we have demonstrated in our preliminary studies that the Alb- Flt3L fusion protein maintains its bioactivity in activating DCs in vitro, and accumulates in the draining lymph nodetoafargreaterextentthanFlt3Lfollowingadministrationinvivo.TheAlb-Flt3Lfusionsafelyproducesmore effective DC expansion and differentiation, and subsequently, upon co-administration of a protein antigen, strongerCD8+andCD4+TcellsandIgG2aantibodyresponses.Wereasonedthatduetoitsabilitytoenhance both T cell- and B cell-mediated immune responses, Alb-Flt3L is a promising adjuvant to enhance both the therapeuticandprophylacticimmunogenicityofTA-CINHPVproteinvaccine.Inthisapplication,wewillevaluate theabilityofAlb-Flt3Lco-administrationwithTA-CINtoenhanceHPVantigen-specificCD8+Tcell,CD4+Tcell, andantibodyresponseselicitedbyvaccination,ascomparedtoFlt3Lco-administration.Wewillfurtherassess how the Alb-Flt3L co-administration with TA-CIN vaccination impacts therapeutic antitumor efficacy against a preclinical HPV16+ cervical cancer model, as well as protection against intravaginal HPV16 virus challenge. Furthermore, we will characterize the mechanisms by which Alb-Flt3L exerts its immunomodulating effects on TA-CIN vaccination. We foresee that the completion of this project will demonstrate potent immunogenicity of theTA-CIN+Alb-Flt3Lvaccination,whichwillsupportourfutureclinicaldevelopmentofthisvaccinationregimen in HPV+ patients and encourage the utilization of Alb-Flt3L to enhance the immunogenicity of other cancer vaccines.